1. Field of the Invention
The present invention relates to an analysis chip comprising a reference range, to a method of analysis of a sample using said chip, and to a diagnostic or analytical kit containing said chip.
The present invention is suitable for the qualitative and especially quantitative analysis of any analyte present in a sample.
The present invention is therefore positioned notably in the area of chemical or biological chips, for example DNA chips, protein chips, antibody chips, antigen chips, cell chips, receptor chips, or chips for other ligands known to a person skilled in the art and capable of specifically capturing and immobilizing one or more analytes.
Usually, applications using DNA chips are divided into two broad categories: gene expression (i) and genotyping (ii).
(i) Gene expression comprises using a DNA chip or chips for studying the variation of all or part of the transcriptome of a cell according to certain biological or physiological parameters: evolution of a cell during the development of an organism, carcinogenesis in a tissue, etc. It is becoming more and more evident that such expression profiles can also serve as diagnostic markers for multifactorial diseases, notably in oncology.(ii) In applications of the genotyping type, the response required from the DNA chip is generally of the qualitative, yes/no type. In fact, we wish to know whether one or more particular sequence(s) is (are) present in the sample. When there is a great need for specificity, for example in the case of the detection of mutations, several oligonucleotides which only differ by one or two bases, are placed on the chip, and generally, a conclusion is made about the presence of the sequence corresponding to the strongest signal, e.g. of fluorescence.
The present invention is suitable for these two broad categories of applications and advantageously solves the many problems of the prior art that are described below, in particular in the carrying out of such studies and profiles.
Owing to its reference range, the chip of the present invention makes it possible to convert detection and/or analysis signals from a chip, for example from a biological chip, to an absolute, reproducible, stable unit that is comparable to other measurement results obtained from the same conversion.
2. Description of the Background Art
For the quantification of variations in gene expression, the biologist nearly always uses two samples which are hybridized simultaneously on a DNA chip: a reference sample, containing a certain mass of RNA or cDNA labelled with a first fluorochrome, for example fluorescein (read in the green), and a sample of interest for which measurement has to be carried out, containing the same mass of RNA or cDNA labelled with a second fluorochrome, for example Cy3 or Cy5 (read in the orange or the red). After hybridization, the DNA chip is read at the two wavelengths, and it is the ratio between the two intensities of the signals emitted which serves as the signal.
It is therefore a question of a relative measurement with internal reference in each experiment. This method works well, but is quite laborious in use, and there are certain pitfalls, such as bias in labelling according to the sequences, or a deviation in reading that varies depending on the wavelength.
Another technique of quantification is used for the gene expression chips developed by the company Affymetrix. In this method, each sequence placed on the chip is represented by a set of oligonucleotides, and each oligonucleotide is in the form of two oligonucleotides, the one perfectly complementary to the required sequence, the other having a point mutation (“mismatch”). The sample of interest is then hybridized on the chip, a single color being used. The complementary-mismatch difference is then used as primary signal, these signals being averaged over the set of oligonucleotides representing a gene. Moreover, a “reference gene” is arranged on the chip by the same technique; this reference gene, taken from the so-called housekeeping genes of the organism, is assumed to maintain constant expression regardless of the physiological conditions of the cell. Then the “primary signal” of each gene is divided by that of the reference gene, for example to avoid the variations in brightness of the fluorescence due to the surroundings. It is then possible for different experiments to be compared with each other.
This technique has the advantage of being monochrome, and therefore does not have the aforementioned drawbacks with the two markers. However, it necessarily uses short oligonucleotides (20 to 25 bases), and therefore there are other pitfalls, since some genes occur in various forms (alternative splicing). This method therefore requires more spots on the DNA chip and more knowledge about the various forms of the RNAs of the genes represented on the chip than with the other methods of the prior art. Moreover, the results vary depending on the choice of reference gene, even when the latter is selected very carefully. Therefore the results cannot always be compared.
The aforementioned methods, provided certain controls and some repetitions of the experiments are employed, can be relatively reliable and therefore offer a research tool that can be used by biologists. However, it must be noted that the measurements are relative, since the signal is a dimensionless number resulting from the ratio between two measurements with units that are generally arbitrary (arbitrary units of fluorescence). Therefore it is not possible to compare experiments performed with different reference samples for example, without additional testing. Moreover, as the measurements are in arbitrary units, it is also rather difficult to know what causes the problems in the case of low intensities: it is difficult to know whether it is an instrumental problem, a problem connected with the chip itself, with the sample, with the operating procedure, or with the result of the experiment.
In applications of the genotyping type, the response required from the DNA chip is generally of the qualitative, yes/no type, as explained above. Furthermore, with the chips of the prior art, the results are relative, since the intensities of different oligonucleotides under investigation on one and the same chip are being compared.
However, for certain applications, such as the detection of mixtures of several sequences, it would be advantageous to be able to compare the signals between several experiments, which is impossible at present with the chips currently available; it is constantly necessary to repeat one or more experiments to obtain relative measurements from which conclusions can be drawn, which is time-consuming and expensive.
Moreover, in routine diagnosis, the problem of quantification of the results can be solved with a calibration curve. A reference sample is diluted in a predetermined manner at various concentrations in a suitable diluent, and the biological test is performed for each dilution away from the chip. This provides a reference curve. When a real biological test is carried out, the result in arbitrary unit is plotted on the reference curve, and the concentration of analyte in the sample is deduced from this. However, it is important for the test to be performed with exactly the same elements as those used for constructing the reference curve. Moreover, in tests with enzymatic development for reading, for example ELISA, ELOSA, etc., a reference curve is also required for each manufacturing batch of the tests. Moreover, additional calibration points for resetting the reference curve for each user must be performed for correcting variations from one machine to another, from one temperature to another, from one laboratory to another, for possible aging of the reagents, etc. These additional calibration tests must be repeated regularly by the users, for example every week.
This method of quantification, although usual, is therefore complicated and expensive. Moreover, it is not easily applicable to DNA chips; in fact, it would be necessary to use a DNA chip for each point of the reference range, and keep repeating the experiment (at each change of batch of marker, or at regular intervals, for example every month, to check for possible drift of the instrumentation), which is too expensive for the laboratories.
Therefore there is a real need for a chip and a method of analysis which enable the many aforementioned problems of the prior art to be solved, and notably which are reliable, accurate, and reproducible; which can be applied to the various known and future DNA chips; which make it possible to compare the signals between several experiments; which make it possible to avoid using a DNA chip for each point of the reference range; and which make it possible to avoid constantly repeating the calibration test (at each change of batch of marker, or at regular intervals, for example every month, and to check for possible drift of the instrumentation), so as to reduce the complexity and costs of analyses on chips, for example on biological chips, for the laboratories, in industry and in research.
In the following description, the references in square brackets [ ] refer to the list of references given after the examples of application of the invention described below.